Laboratory evaluation of a new integrative assay to phenotype plasma fibrinolytic system.

BACKGROUND: There is currently no universal and standardized test available to phenotype plasma fibrinolytic system. AIMS: Our main aims were to evaluate the performances of the 'global fibrinolysis capacity' assay (GFC) performed with the Lysis Timer® instrument, and to study the influence of some preanalytical conditions. METHOD: Euglobulin clot lysis time (ECLT) and GFC were performed under several preanalytical conditions. RESULTS: GFC showed satisfactory intra- and inter-run precision. Frozen controls and reagents showed stability over the studied period. There was no statistically significant difference between GFC assessed in plasma samples processed at 4 °C or at 20 °C. GFC assessed with frozen-thawed plasma samples was prolonged when compared to fresh samples (p = 0.014). The centrifugation scheme had no influence on PAI-1 activity levels, GFC and ECLT. Reference interval for GFC ranges from 29.3 (C I90% = 26.9-31.9) to 49.5 (90% CI = 45.9-52.2) minutes. In addition, a preliminary study in 40 healthy volunteers and 43 adult patients referred for investigation of a bleeding disorder was conducted to compare GFC and ECLT assays in their ability to classify samples with shortened or prolonged clot lysis times. Disagreements between ECLT and GFC were observed for 23 samples (out of 83), most of them minor. CONCLUSION: GFC is suitable and convenient for a broad clinical use and can be performed with frozen-thawed plasma samples. Unlike ECLT, GFC is designed to take into account the balance between inhibitors and activators of the fibrinolytic system and could detect both hypo- and hyperfibrinolytic states. Whether it is as suitable as or even better than ECLT to detect a bleeding tendency due to a hyperactive fibrinolytic system deserves to be properly investigated.

Perls' Stain Guidelines from the French-Speaking Cellular Hematology Group (GFHC).

In order to standardize cellular hematology practices, the French-speaking Cellular Hematology Group (Groupe Francophone d'Hématologie Cellulaire, GFHC) focused on Perls' stain. A national survey was carried out, leading to the proposal of recommendations on insoluble iron detection and quantification in bone marrow. The criteria presented here met with a "strong professional agreement" and follow the suggestions of the World Health Organization's classification of hematological malignancies.

Pseudothrombocytopenia-A Review on Causes, Occurrence and Clinical Implications.

Pseudothrombocytopenia (PTCP), a relative common finding in clinical laboratories, can lead to diagnostic errors, overtreatment, and further (even invasive) unnecessary testing. Clinical consequences with potential life-threatening events (e.g., unnecessary platelet transfusion, inappropriate treatment including splenectomy or corticosteroids) are still observed when PTCP is not readily detected. The phenomenon is even more complex when occurring with different anticoagulants. In this review we present a case of multi-anticoagulant PTCP, where we studied different parameters including temperature, amikacin supplementation, measurement methods, and type of anticoagulant. Prevalence, clinical risk factors, pre-analytical and analytical factors, along with clinical implications, will be discussed. The detection of an anticoagulant-dependent PTCP does not necessarily imply the presence of specific disorders. Conversely, the incidence of PTCP seems higher in patients receiving low molecular weight heparin, during hospitalization, or in men aged 50 years or older. New analytical technologies, such as fluorescence or optical platelet counting, will be soon overturning traditional algorithms and represent valuable diagnostic aids. A practical laboratory approach, based on current knowledge of PTCP, is finally proposed for overcoming spuriously low platelet counts.

Prognostic value of high-sensitivity measurable residual disease assessment after front-line chemoimmunotherapy in chronic lymphocytic leukemia.

Measurable residual disease (MRD) status is widely adopted in clinical trials in patients with chronic lymphocytic leukemia (CLL). Findings from FILO group trials (CLL2007FMP, CLL2007SA, CLL2010FMP) enabled investigation of the prognostic value of high-sensitivity (0.7 × 10-5) MRD assessment using flow cytometry, in blood (N = 401) and bone marrow (N = 339), after fludarabine, cyclophosphamide, and rituximab (FCR)-based chemoimmunotherapy in a homogeneous population with long follow-up (median 49.5 months). Addition of low-level positive MRD < 0.01% to MRD ≥ 0.01% increased the proportion of cases with positive MRD in blood by 39% and in bone marrow by 27%. Compared to low-level positive MRD < 0.01%, undetectable MRD was associated with significantly longer progression-free survival (PFS) when using blood (72.2 versus 42.7 months; hazard ratio 0.40, p = 0.0003), but not when using bone marrow. Upon further stratification, positive blood MRD at any level, compared to undetectable blood MRD, was associated with shorter PFS irrespective of clinical complete or partial remission, and a lower 5-year PFS rate irrespective of IGHV-mutated or -unmutated status (all p < 0.05). In conclusion, high-sensitivity (0.0007%) MRD assessment in blood yielded additional prognostic information beyond the current standard sensitivity (0.01%). Our approach provides a model for future determination of the optimal MRD investigative strategy for any regimen.

[Management of the thrombotic risk associated with COVID-19: what is the role of the hemostasis laboratory?] / Apport du laboratoire d'hémostase dans la gestion du risque thrombotique associé au Covid-19.

COVID-19 is associated with disturbances of hemostasis in the laboratory and an increased thrombotic risk. Routine laboratory tests - activated partial thromboplastin time (aPTT), prothrombin time, Clauss fibrinogen and D-dimers levels measurement - are used for the evaluation of the thrombotic risk and the monitoring of hemostasis, but are subject to several drawbacks that may affect the reliability and clinical relevance of the delivered results. Another challenge for the hemostasis laboratory is the monitoring of heparin treatment. For instance, the issue of the monitoring of unfractionated heparin remains debated, the more so when there is a tremendous inflammatory response. This brief review considers the role of laboratory tests of hemostasis in the management of COVID-19 and discusses their main limitations to be kept in mind.

Effects of Time-Interval since Blood Draw and of Anticoagulation on Platelet Testing (Count, Indices and Impedance Aggregometry): A Systematic Study with Blood from Healthy Volunteers.

Platelet count, indices (mean volume, young-immature platelet fraction) and aggregation are widely used laboratory parameters to investigate primary hemostasis. We performed a systematic, thorough evaluation of the influence of the time-interval since blood draw from 20 healthy individuals and of the anticoagulation of collected blood on such parameters. Blood was anticoagulated with citrate, K2-ethylenediaminetetraacetic acid (EDTA) and hirudin and analyzed 5, 30, 60, 120 and 180 min after blood draw. Multiple electrode aggregometry (MEA) was performed with either hirudin (half-diluted with NaCl) or citrate samples (half-diluted with NaCl or CaCl2 3 mM). Platelet count and indices (Sysmex XN-20) were rather stable over time with EDTA blood. MEA results were lower with citrate blood than with hirudin blood; supplementation with calcium was partially compensatory. MEA results were also lower when performed less than 30 or more than 120 min after blood draw. Platelet clumping, quantitatively estimated with microscope examination of blood smears, was more important in hirudin blood than citrate or EDTA blood and could explain some of the differences observed between preanalytical variables. The results stress once more the importance of preanalytical variables in hemostasis laboratory testing. Decision thresholds based on those tests are only applicable within specific preanalytical conditions.

Evaluation of a new thromboplastin reagent STA-NeoPTimal on a STA R Max analyzer for the measurement of prothrombin time, international normalized ratio and extrinsic factor levels.

INTRODUCTION: We aimed at evaluating the performance of a new prothrombin time (PT) reagent (STA-NeoPTimal) with two other PT reagents (STA-Neoplastine R and STA-Neoplastine CI Plus) and the reference PT reagent used in our laboratory (ReadiPlasTin). METHODS: Evaluation consisted in intra- and interassay precision assessment, determination of sensitivity to unfractionated heparin (UFH) or enoxaparin in spiked samples and to direct oral anticoagulants (DOACs) in patients (n = 43). Method comparison of the 4 PT reagents, factor II, V, VII and X assays was tested on normal (n = 20) and abnormal samples: VKA (n = 47), preoperative (n = 23), liver failure (n = 12) and burned patients (n = 37). RESULTS: Analytical performance met manufacturers' criteria for all reagents. All PT reagents gave correlation coefficients >0.8 and even >0.9 in many situations. In some VKA samples, differences ≥ 0.5 INR units were found in samples within and above therapeutic ranges. For burned patients, PT correlations were good but with some minimal bias (<5.0%) while factor assays gave very consistent results (R > .8 and mainly >0.9). As expected, poor responsiveness of the PT to DOAC concentrations was observed with all four assays. CONCLUSION: The STA-NeoPTimal showed comparable performance to ReadiPlasTin, making it suitable for VKA control, detection of factors II, V, VII, X deficiency and assessment of liver disease coagulopathy. However, for patients receiving VKA, some significant differences were observed. We confirmed the inability of the PT assay to detect residual DOAC concentrations. Finally, burned patients results showed that recombinant thromboplastins were less sensitive to factor deficiencies in comparison to extraction thromboplastins.

Clustering and Kernel Density Estimation for Assessment of Measurable Residual Disease by Flow Cytometry.

Standardization, data mining techniques, and comparison to normality are changing the landscape of multiparameter flow cytometry in clinical hematology. On the basis of these principles, a strategy was developed for measurable residual disease (MRD) assessment. Herein, suspicious cell clusters are first identified at diagnosis using a clustering algorithm. Subsequently, automated multidimensional spaces, named "Clouds", are created around these clusters on the basis of density calculations. This step identifies the immunophenotypic pattern of the suspicious cell clusters. Thereafter, using reference samples, the "Abnormality Ratio" (AR) of each Cloud is calculated, and major malignant Clouds are retained, known as "Leukemic Clouds" (L-Clouds). In follow-up samples, MRD is identified when more cells fall into a patient's L-Cloud compared to reference samples (AR concept). This workflow was applied on simulated data and real-life leukemia flow cytometry data. On simulated data, strong patient-dependent positive correlation (R2 = 1) was observed between the AR and spiked-in leukemia cells. On real patient data, AR kinetics was in line with the clinical evolution for five out of six patients. In conclusion, we present a convenient flow cytometry data analysis approach for the follow-up of hematological malignancies. Further evaluation and validation on more patient samples and different flow cytometry panels is required before implementation in clinical practice.

Platelet Counting: Ugly Traps and Good Advice. Proposals from the French-Speaking Cellular Hematology Group (GFHC).

Despite the ongoing development of automated hematology analyzers to optimize complete blood count results, platelet count still suffers from pre-analytical or analytical pitfalls, including EDTA-induced pseudothrombocytopenia. Although most of these interferences are widely known, laboratory practices remain highly heterogeneous. In order to harmonize and standardize cellular hematology practices, the French-speaking Cellular Hematology Group (GFHC) wants to focus on interferences that could affect the platelet count and to detail the verification steps with minimal recommendations, taking into account the different technologies employed nowadays. The conclusions of the GFHC presented here met with a "strong professional agreement" and are explained with their rationale to define the course of actions, in case thrombocytopenia or thrombocytosis is detected. They are proposed as minimum recommendations to be used by each specialist in laboratory medicine who remains free to use more restrictive guidelines based on the patient's condition.

Two-site evaluation of a new workflow for the detection of malignant cells on the Sysmex XN-1000 body fluid analyzer.

INTRODUCTION: The presence of high fluorescent cells (HF-BF) on the Sysmex XN-1000 hematology analyzers has gained interest regarding the prediction of malignant cells in body fluids, but lacks sensitivity. We aimed to increase this sensitivity by combining HF-BF value, automated results, and clinical information. METHODS: We evaluated a new workflow for the management of body fluids in the hematology laboratory, including the HF-BF criterion and clinical information. In two laboratories, 1623 serous fluids were retrospectively analyzed on the XN-1000 BF mode. All samples were morphologically screened for malignant cells. Optimal HF-BF cutoffs were determined to predict their presence. Thereafter, the added value of clinical information was evaluated. Other reflex testing rules (eosinophilic count >5% and presence of the WBC Abnormal Scattergram flag) were also used to refine our workflow. RESULTS: Optimal HF-BF cutoffs in the two hematology centers were 108 and 45 cells/µL, yielding a sensitivity/specificity of 66.7/93.6% and 86.8/66.6% for malignant cell detection. When adding clinical information, sensitivity/specificity evolved to 100.0/68.9% and 100.0%/not determined. Of 104 samples containing malignant cells, 97 had positive clinical information; the remainder had a HF-BF > cutoff. CONCLUSION: Combining clinical information and HF-BF reached 100% sensitivity for malignant cell detection in body fluid analysis. Lack of robustness of the optimal HF-BF cutoff deserves the use of local cutoffs. Rapid automated results reporting from the XN-1000 BF mode are also feasible in clinical practice. Prospective evaluation of the workflow is needed before its implementation in clinical practice.

Prospective and comparative study of paroxysmal nocturnal hemoglobinuria patients treated or not by eculizumab: Focus on platelet extracellular vesicles.

Thrombosis are severe complications of paroxysmal nocturnal hemoglobinuria (PNH), effectively reduced by eculizumab. Extracellular vesicles (EVs) may play a central role. The objective of this study was to assess the procoagulant activity of plasma isolated from PNH patients (treated or not by eculizumab) and to quantify their circulating EVs.We iteratively collected the platelet-free-plasma of 17 PNH patients and 16 matched healthy volunteers, quantified their circulating EVs by flow cytometry and evaluated their procoagulant activity by thrombin generation and STA-Procoag-procoagulant phospholipid (PPL) assays.A significant decrease of EVs from platelets (P = .024) and an increase of the STA-Procoag-PPL clotting time (P = .049) was observed after initiation of eculizumab and up to 11 weeks after. This reduction of prothrombotic biomarkers was not observed with the thrombin generation test due to a lack of sensitivity of this assay. Active hemolysis was observed in 90% of patients and elevated D-dimers in 41% of them. However, no significant difference was observed between patients and control subjects regarding the procoagulant activity, the EVs quantity, or the cellular origin. Lactate dehydrogenase (LDH) levels were lower in eculizumab-treated patients compared to nontreated patients (441 vs 2448 IU/L). D-dimers and LDH decreased after administration of eculizumab (mean decrease of 1307 ng/mL and 4159 IU/L, respectively).These observations suggest a decrease of the phospholipid-dependent procoagulant potential of EVs after eculizumab therapy in PNH patients. TRIAL REGISTRATION:: NUB: B039201214365.

Extracellular Vesicles in Red Blood Cell Concentrates: An Overview.

Red blood cell (RBC) concentrates may be stored for up to 42 days before transfusion to a patient. During storage extracellular vesicles (EVs) develop and can be detected in significant amounts in RBC concentrates. The concentration of EVs is affected by component preparation methods, storage solutions, and inter-donor variation. Laboratory investigations have focused on the effect of EVs on in vitro assays of thrombin generation and immune responses. Assays for EVs in RBC concentrates are not standardized. The aims of this review are to describe the factors that determine the presence of erythrocyte-EVs in RBC concentrates, the current techniques used to characterize them, and the potential role of EV analysis as a quality control maker for RBC storage.

Diagnosis and management of PNH: Review and recommendations from a Belgian expert panel.

Despite its considerable morbidity and mortality, paroxysmal nocturnal haemoglobinuria (PNH) is still underdiagnosed. Patients with PNH can suffer from cardiovascular, gastrointestinal, neurological or haematological symptoms and refer to several specialists. The aim of this paper is to review the diagnosis and the management of PNH patients, with the primary focus on identifying high-risk groups. Additionally, the implementation and prognostic value of the defined high-risk groups will be commented on and the management of PNH patients is discussed from a Belgian perspective. Finally, based on the available data, recommendations are provided. Eculizumab is a potent C5 complement inhibitor and reduces intravascular haemolysis and thrombosis in PNH patients and improves their quality of life. As thrombosis is the main cause of death in PNH patients, identifying high-risk PNH patients in need of therapy is essential. Currently, novel complement inhibitors are in development and the first data seem promising. Another challenge in PNH is to identify new markers to assess the thrombotic risk to achieve a better risk-based prophylactic anti-thrombotic management. Finally, because of the low prevalence of the disease, PNH patients should be included in the prospective PNH registry, which will offer new insights on the natural course of the disease and the impact of treatment of PNH.

Betrixaban: Impact on Routine and Specific Coagulation Assays-A Practical Laboratory Guide.

INTRODUCTION: Betrixaban is a novel direct oral factor Xa inhibitor approved by the Food and Drug Administration for prophylaxis of venous thromboembolism in adult patients hospitalized for an acute illness at risk for thromboembolic complications. Assessment of the anti-coagulant effect of betrixaban may be useful in some situations. Also, clinicians need to know how routine coagulation assays are influenced. OBJECTIVE: The aim of this study is to determine which coagulation assay(s) should be used to assess the impact of betrixaban on haemostasis and provide laboratory guidance for their interpretation. MATERIALS AND METHODS: Betrixaban was spiked at final concentrations ranging from 0 to 250 ng/mL in platelet-poor plasma. Different reagents from several manufacturers were tested and the impact of betrixaban on pro-thrombin time (PT), activated partial thromboplastin time (aPTT), dilute Russel viper venom time (dRVV-T), chromogenic anti-Xa assays, thrombin generation assay (TGA), and a large panel of haemostasis diagnostic tests has been assessed. RESULTS: A concentration-dependent prolongation of aPTT, PT and dRVV-T is observed. The sensitivity mainly depends on the reagent. Chromogenic anti-Xa assays show high sensitivity depending on the reagent and/or the methodology. These assays applicable for other direct factor Xa inhibitors have to be adapted to obtain a relevant range of measurement. TGA may also be attractive to assess the anti-coagulant activity of betrixaban. CONCLUSION: Adapted chromogenic anti-Xa assays are the most appropriate assays to estimate the concentration of betrixaban. Betrixaban significantly affects several haemostasis diagnostic tests and this needs to be taken into consideration when requesting and interpreting such tests.